1.1 Field of the Invention
The present invention relates generally to the fields of sensor technology and molecular diagnostics. More particularly, it concerns the quantitative electrochemical detection of selected nucleic acid sequences in complex mixtures.
1.2 Description of Related Art
The determination of a specific DNA or RNA target nucleic acid sequence or segment present in air, food, water, environmental or clinical samples is of great significance in the medical, microbiology, food and water safety-testing, and environmental monitoring fields. The detection of the presence of a DNA or RNA sequence in a sample can rapidly and unambiguously identify genetic defects, oncogenic events and bacterial, viral or parasitic agents of concern.
Diagnosis of numerous infectious and inherited human diseases is possible with clinical assays that detect known DNA sequences characteristic of a particular disease (Molecular Diagnostics, 1993; Benn et al., 1987; Lowe, 1986). Unfortunately, few detection methods are suitable for routine diagnostic use either in the clinical laboratory or in the field setting. Many assays are not sufficiently rapid, inexpensive, simple or robust for routine application.
Recent advances in molecular diagnostics have focused on methods of detection at the genetic level. Since the advent of PCR(trademark) technology, the ability to detect or identify point mutations, allelic variation, the presence of minute amounts of a pathogen, species, and from individual microscopic samples, to give a few examples, has been vastly improved. Yet even with the advances of PCR(trademark) technology many limitations still exist which prevent diagnostic assays from being as versatile as desired or needed.
For example, one of the biggest problems with highly sensitive assays, such as PCR(trademark) based assays, is contamination by extraneous air-borne organisms or by human contact. In general, if a molecular diagnostic assay is highly sensitive and can detect minute quantities of a selected or target nucleic acid segment, the sample to be assayed must be highly purified or at least not contain extraneous nucleic acid fragments which may be at least partially complementary to the target nucleic acid segment. Otherwise, false positive or ambiguous readings may result. Of course, obtaining highly purified samples is not cost ineffective due to time and labor involved. Strong technical expertise and well-equipped diagnostic laboratories are required in most cases. Thus in many instances where a highly sensitive assay is desirable, it is impractical, if not impossible, to perform such assays.
A variety of methods have been used to detect and quantitate polynucleotide sequences. Almost all of these begin with amplification of individual sequences or their transcripts by PCR. Some require separation by gel electrophoresis and/or laser detection. These needs greatly complicate the development of good technology for scale-up, automation, and reduction of cost. Probe-array technologies are being developed by a number of companiesxe2x80x94including Nanogen (San Diego, Calif.) and Affymetrix (Santa Clara, Calif.). The electrochemical methods described do not rely upon electrophoresis or laser technology, and can make use of DNA or PNA probes in arrays.
In cases where it is desirable to detect more than one target nucleic acid species in a sample, or the sample is highly complex, highly sensitive assays must be customized to detect the desired targets. Although not entirely understood, it is well known that many highly sensitive assays suffer from undue interference caused by background sample material. In fact, the time and labor required to develop some assays is so great that the assays are not useful and less sensitive means of analysis are employed.
Diagnostic assays that do not require high purity samples are less sensitive and therefore cannot detect minute amounts of target. Thus larger or more concentrated samples must be obtained. In some cases, larger sample quantities are not available or are too costly.
Probe assays, including oligonucleotide-probe and gene-probe assays, have been developed recently in an attempt to take advantage of the ability to detect selected DNA or RNA sequences with high sensitivity and replace conventional detection methods. However, these techniques still tend to be labor-intensive and often require significant technical training and expertise. Further, highly sensitive gene-probe assays require specialized equipment and are generally not compatible with field settings. Those that can be used in a field setting are limited to determining the presence or absence of a target nucleic acid fragment and do not provide quantitative information. The utility of gene-probe assays for environmental monitoring and other uses outside of a laboratory setting is limited.
Moreover, gene-probe assays may use a label that is either toxic or requires substantial expertise and labor to use. Radiolabeling is one of the most commonly used techniques because of the high sensitivity of radiolabels. But the use of radiolabeled probes is expensive and requires complex, time consuming, sample preparation and analysis and special disposal. Alternatives to radioactivity for labeling probes include chemiluminescence, fluorimetric and colorimetric labels (Kricka, 1993) but each alternative has distinct disadvantages. Colorimetry is relatively insensitive and has limited utility where minute amounts of sample can be obtained. Samples must also be optically transparent. Fluorimetry requires relatively sophisticated equipment and procedures not readily adapted to routine use. Chemiluminescence, although versatile and sensitive when used for Southern blots, northern blots, colony/plaques lift, DNA foot-printing and nucleic acid sequencing, is expensive, and is not well-adapted for routine analysis in the clinical laboratory.
Another limitation to the versatility of oligonucleotide-probe assays is that virtually all current oligonucleotide-probes are designed as heterogeneous assays, i.e., a solid phase support is used to immobilize the target nucleic acid so that free, non-hybridized probe can be removed by washing. Complex procedures and long incubation times (one to several days) are usually required which makes these assays difficult to incorporate into the simple and rapid formats that are desirable for clinical applications or on-site analysis (Molecular Diagnostics, 1993).
Various techniques exist for the detection of differential gene expression into closely matched cell populations. These include substractive cloning (Sagerstrom et al., 1997, Differential Display (Liang and Pardee, 1992), serial analysis of gene expression (SAGE) (Velculescu et al, 1995), expressed sequence tags (ESTs) (Adams et al. 1991). While all of the above-mentioned approaches have yielded significant results most have drawbacks. Substractive cloning, SAGE and ESTs tend to be labor intensive and costly and therefore not suitable for use on a routine basis. For example SAGE and ESTs entail the use of hundreds or thousands of DNA sequencing reactions. Recently the feasibility of hybridization based technology as well as its superiority to other screening process has been shown. The human genome project has identified new genes and unique ESTs at a rapid rate. As of Nov. 30, 1998 17,583 human genes or complete coding sequences had been identified and 52,277 unique ESTs had been cataloged. DNA microarray or gene chip technology has catalyzed further advances. The technology involves the positioning of highly condensed and ordered arrays of DNA probes on glass slides or nylon membranes. Up to 50,000 DNA fragments, each representing an individual gene can be placed on a single glass slide and up to 5,000 placed on a nylon membrane. The resulting microarrays can then be used to examine presence and levels of gene expression.
Alternatives to gene-probe and other assay methods of detecting nucleic acid sequences have employed electrochemical biosensors that employ intercalators and discriminate between immobilized single-stranded and double-stranded DNA (Hashimoto et al., 1994; Millan et al., 1994; Millan and Mikkelsen, 1993). While such biosensors are capable of detecting a known target DNA sequence, they are handicapped by the fact that the electrode must be cleaned between each use. The procedures used to strip away the hybridized target DNA from the electrode surface are not suitable for widespread screening applications, such as clinical diagnostics Where labor and expense must be kept minimal and speed is essential, or in settings outside of the laboratory such as field testing.
DNA diagnostics have recently achieved importance because of the advances in molecular biology that have highlighted the importance of gene mutations and hereditary diseases. Many studies have focused on the breast cancer susceptibility gene BRCA1 which is estimated to account for a large fraction of hereditary breast cancer and the majority of familial breast/ovarian cancer. Over 111 unique BRCA1 mutations distributed throughout the gene have been described (Shatkuck-Eldens et al., 1995: Breast Cancer Information Core Database). Many methods have been used to screen for BRCA1 mutations. Almost all of these based on amplification of individual exons or their transcripts by PCR(trademark) (Nollau and Wagener, 1997). All require separation by gel-electrophoresis. The need for electrophoresis greatly complicates scale-up automation and streamlining of procedures. A method that does not require electrophoresis (Affymetrix, Santa Clara, Calif.) is epifluorescence confocal scanning microscopy that utilizes high-density oligonucleotide arrays of over 96,000 oligonucleotides to scan 3,450 bases of exon 11 of BRCA1 (Hacia et al., 1996). Although it is powerful technique it is expensive and not readily available to most laboratories.
There are many human diseases associated with known gene alterations. These diseases include cystic fibrosis, muscular dystrophy, sickle cell anemia, phenylketonuria, thalassemia, hemophelia, xcex11-antitrypsin deficiency and lipoprotein metabolism disorders (Ben et al., 1987; Lowe, 1986; Landegren et al., 1988; Young et al., 1989; Kricka, 1993). Quantitative analysis of human genes is also useful for analysis of amplified oncogenes (Altitalo, 1987) and in a measurement of gene expression levels in tumors (Slamon et al., 1989). Genetic aspects of human diseases contributed by factors such as BRCA1, BRCA2 and p53 mutations all of which confer high cancer risk would benefit from gene analysis as would mutational changes in genes caused by chemical radiation.
Breast cancer is considered to have a hereditary cancer risk component. Among women who have a blood relative with the disease the risk of developing breast cancer is 1 in 5. Two mutated genes increase a women""s chances of developing breast cancer and genetic tests for detecting women at greatest risk have been used clinically.
Several genes in have been associated with the pre-disposition to cancer. These include BRCA1, p53, RB1 and APC which are just a few of the more than 20 genes identified that are associated with the pre-disposition to cancer (Fearon, 1997; Ponder, 1997). The BRCA1 gene and its expressed protein as it relates to diagnosis and potential treatment of disease, has utility in diagnosis and potential disease treatments.
Clearly, there is a need for improved detection of nucleic acid sequences. Unfortunately, few assays are currently available for routine monitoring and/or diagnostic use because of the expense, complexity and/or physical limitations which prevent their use outside of a well-equipped laboratory. As discussed, the few existing assays that have limited applications and do not meet the diverse needs of clinical diagnostics and field testing.
The present invention seeks to address these and other deficiencies inherent in the prior art by providing simple and sensitive electrochemical methods of detecting virtually any type of nucleic acid sequence or segment, provided the target nucleic acid has been identified. The target nucleic acid sequences are detectable in synthetic and natural environments by capturing target nucleic acid sequences at the surface of an electrochemical sensor through hybridization with specific nucleic acid probes and subsequent triggering of an electrochemical reaction that generates current.
In preferred embodiments, the invention comprises a biosensor array having a plurality of both working and reference electrodes formed on a circuit board. The working electrodes are linked or attached to bioreporter molecules that are typically a protein or a nucleic acid, including peptides, polypeptides and peptide nucleic acids. The proteins may be antigens or antibodies, protein variants or functional derivatives. Virtually any protein or peptide may be used as a bioreporter, including enzymes with a variety of functions, e.g., reductases, peroxidases or phosphatases. The nucleic acids may be oligonucleotides, such as a DNA or RNA, including mRNAs, rRNAs and tRNAs.
In a particularly preferred embodiment, the plurality of working and reference electrodes of the biosensor array are comprised on a printed circuit board, preferably screen printed, with a plurality of labeled nucleic acid segments are attached to the surface of the working electrodes. The labeled segments will generate an electric current when an electric potential is applied to the working electrode after the attached labeled nucleic acid segments are hybridized or annealed with a target nucleic acid. Of course a plurality of labeled proteins, peptides, polypeptides or protein nucleic acids may likewise be attached to the working electrode and will produce an electric signal under similar conditions subsequent to binding or combining with a selected substrate or receptor. The attached nucleic acids or other molecules attached to the working electrode need not be identical and each different labeled nucleic acid may, for example, bind to a different target nucleic acid.
An electric potential applied to the working electrodes may be applied with a multiplexed potentiostat. This allows measurement of any current produced on binding of the attached nucleic acid or other substance binding with a target molecule in situations where each labeled nucleic acid is hybridized to a different target and each generates a current.
The biosensor electrodes will be connected or operably linked to an electrochemical pulse analyzer which provides an electrical potential to the working electrode and also detects any signal produced by the bioreporter molecule as a result of sending an electrical pulse across the working electrode. The electrical pulse may be provided by a potentiostat may generate a current from the bioreporter under conditions where the bioreporter combines with a target molecule.
The working electrodes may be carbon or metal electrodes, including gold, silver, platinum, iridium mercury, nickel, copper, palladium or colloidal forms of these metals such as colloidal gold, colloidal silver, colloidal carbon or combinations of these materials. The particular electrodes will be chosen to some extent for their ablility to attach a selected reporter. In certain cases it will be advantageous to coat the electrode surface with a material that will increase binding of a reporter molecule. Typical surface coatings on gold or colloidal gold, for example, include avidin, streptavidin, protein G, protein A and NeutrAvidin. A preferred working electrode is a gold or colloidal gold electrode surface coated with biotin, digoxigenin or NeutrAvidin that attaches to a short nucleic acid segment specifically bounds or hybridized with a species specific region of a DNA or RNA. Nuclei acid segments are conveniently about 15 to about 25 bases in length, e.g. SEQ ID NO:8 and SEQ ID NO:9. The electrodes are useful in detecting plant, animal, and mammalian DNA or RNA and targeted nucleic acid sequences from microbes and vertebrates, including humans.
The type of coating will of course determine the nature of the attachment to a reporter molecule. Generally this will be a link arising from chemical crosslinking, covalent bonding but may also be from charge-charge interaction or adsorption.
The biosensor is preferably small in size for easy handling, and the working electrode may have a surface area from about 0.001 mm2 to about 100 mm2. The array may comprise several sample wells including 4, 96, 384, 1536-well configurations or larger; however, the 96-well array is currently convenient for most applications.
In a most preferred embodiment, the circuits are screen printed. Printed circuit board technology may be employed to photolithograph the circuit onto a board. Where screen printed circuits are employed, a preferred embodiment is screen printed of carbon and silver over the working electrodes in the biosensor array in the area around the sample wells or where the sample is applied to the circuit board.
Bioreporters may be selected to recognize a single target molecule or several different bioreporters may be used in the same array, each specific for a different target molecule. Of course one may wish to utilize several reporters that each recognize the same molecule to assure that the target molecule is detected. Target molecules may be in separate or multiple samples that are applied to the multiple array for analysis.
Target molecules will be selected that can be recognized and will attach to the bioreporter. Depending on the types of molecules, attachment may be by hybridization, annealing, charge-charge interaction, hydrophobic or covalent bonding. Generally, the targets are oligonucleotides, including DNA, mRNA, rRNA, tRNA or PNAs. Of particular interest are the many ESTs available that may be useful as bioreporter targets in identifying new genes. Target molecules will typically include mRNA expressed from any of a number of microorganisms, pathogens and mutant genes. Examples include mRNA from E. coli, Salmonella, Chlamydia, Neisseria, polio virus and Vibrio. The biosensor may also be used to detect mRNA expressed form an APOA1 BRCA1, p53 or sickle cell human xcex2-globin gene. DNA deletions, insertions and single base changes in human and animal genes are also detectable. The source of nucleic acid, whether DNA or RNA, may be from humans, animals or plants.
The biosensor may comprise an apparatus or be used in a system that includes the necessary components for detecting and measuring a signal produced by one or more bioreporters. An apparatus will comprise integrated circuit including the biosensor array combined with a power supply and a detector. Such integrated circuits are known to those of skill in the art. Systems including the biosensor array may additionally include means for measuring an electrochemical signal after a potential is applied across a working electrode. Applying the electrical potential and measuring the electrochemical signal are conveniently accomplished with a programmed processor.
The signal to be detected from the bioreporter is measured by pulse amperometry, preferably by intermittent pulse amperometry or differential pulse amperometry.
Alternatively, the biosensor need not comprise a plurality of working and reference electrodes but may comprise a single working electrode and a single reference electrode. Whether in an array or a single working electrode, the biosensor may optionally include one or more counter electrodes.
The methods and apparatus herein disclosed are particularly suitable for the detection of infectious disease organisms; for example malaria, enterobacteria, viruses such as dengue, hantavirus, encephalitis virus, filovirus as well as Brucella, Clostridium, anthrax and plague causing microorganisms. Additionally, the method is suitable for the rapid detection of altered gene sequences and heterozygus mutations. Assays for breast cancer sequence mutation, point mutations in the Factor V gene, detection of disease-associated mRNAs and detection of low levels of circulating K ras tumor DNA in blood plasma are also within the scope of the invention. Selective detection of the polio virus, hepatitus A virus, rotavirus, and liver-associated mRNA has been achieved with the disclosed method.
An important aspect of the invention is the use of the disclosed biosensor and biosensor arrays and apparatus to detect and identify target molecules. One therefore will select an appropriate bioreporter molecule to attach to one or more working electrodes based on a selected target molecule. The working electrode is comprised within a circuit that will generate a current when an electric signal is applied to the working electrode and after the bioreporter has interacted with the target molecule. Preferably, the electrical signal is a pulsed signal. The resulting current is indicative of the presence of the target molecule and may be quantitatively related to the amount of target molecule detected.
The target molecule may be from any of a variety of sources, including microorganisms, plants or mammals and particularly from pathogens such as E. coli, Salmonella, Chalmydia, Neisseria, polio virus and Vibrio. Preferably, the targets are proteins, peptides, polypeptides or nucleic acids, including DNA, mRNA, tRNA or rRNA. The method readily detects deletions, insertions or single base alterations in DNA This has been exemplified with human BRCA1 and sickle cell human xcex2-globin gene.
Methods for detecting a target nucleic acid sequence may include additional steps to further enhance detection and measurement of the target. A hybridized target nucleic acid, hybridized with the bioreporter (capture) molecule and/or detector probe, may be incubated with precursor DNA or RNA nucleotide bases to extend the length of the hybridized target. This is accomplished with a single primer if desired. Where a reporter and a detector probe are employed, the bioreporter probe may be 3xe2x80x2-end blocked and the detector probe may have a 3xe2x80x2-hydroxyl group. Alternatively, the bioreporter may be extended before hybridization with the target molecule. The precursors may be labeled, for example with fluorescein or other fluorescent label.
Methods of a nucleic acid are another important aspect of the invention using the disclosed system. In this case, one prepares or obtains a capture probe complementary to a first selected region of the target nucleic acid. The capture probe is attached to the surface of a working electrode. A labeled detector probe is obtained and this probe is complementary to a second selected region of the target nucleic acid. The target nucleic acid which may be a DNA or an RNA, is then incubated with the capture and detector probes, separately or together, under conditions that allow or promote hybridization or annealing of the target with both the capture and the detector probes. The incubation step in many cases will be performed at an elevated temperature, preferably at or near the melting point temperature of the target/probe hhybrid.
The amount of target nucleic acid may be extremely low and in such cases amplification may be advisable. One method may then utilize additional steps by generating a PCR-amplified double-stranded nucleic acid with a forward primer and a 5xe2x80x2-phosphate-modified reverse primer positioned outside a selected of a target nucleic acid. The double-stranded nucleic acid molecule will then be digested with an exonuclease, preferably lambda exonuclease, in order to degrade the 5xe2x80x2-phosphate modified strand. This results in a single strand target nucleic acid which is then detected as described. The forward primer may optionally be modified with biotin, or 5xe2x80x2-modified with phosphate and the reverse primer optionally modified with biotin or a functionally equivalent protein.
The method is readily applied to detection of mutations in nucleic acid sequences. In a preferred embodiment, a nucleic acid segment from a genomic DNA sample is amplified and the resulting double-stranded DNA digested with an exonuclease, preferably lambda exonuclease. The resulting single-stranded DNA is hybridized to a labeled detector probe and a labeled capture probe and then attached to the surface of an electrode. The mutation is detected by the electrochemical method described. Mutations in BRCA1 gene and a single base in sickle cell anemia gene have been detected with this method.
An important aspect of the disclosed methods is the use of differential pulse amperometry for the detection and quantitation of a target molecule. This procedure includes applying a first potential to the working electrode. The potential applied is preferably close to or at the open circuit potential. A second shorter potential is then applied to the working electrode to oxidize or reduce any reporter molecules at or near the working electrode surface. The difference between the current measured close to the end of each pulse then can be used to indicate the presence of the hybridized target nucleic acid.
Intermittant pulse amperometry is yet another preferred step in the detection of a target nucleic acid. A pulse of potential is directed to the working electrode to which a target/capure or target/reporter molecule is attached. The reporter or capture molecule is electrochemically oxidized or reduced. The working electrode is then disconnected from the potentiostat circuit for a period at least as long as the applied pulse which may be applied for a period of time from about 0.1 millisecond to about 100 milliseconds. Pulse separation time is from 1 millisecond to about 10 seconds. The measured current generated by each pulse can be related to the amount of target nucleic acid present.
Alternatively, the capture probe may be hybridized to the target molecule in solution, then attached to the working electrode. The detector probe is then hybridized with the hybridized capture/target nucleic acid. The capture probe may be labeled with avidin, streptravidin, NeutrAvidin, protein G , protein A or biotin which attaches to the working electrode surface. The detector probe may be labeled with fluorescein, digoxigenin, horseradish peroxidase, alkaline phosphatase or the like. The electrode surface is typically gold, colloidal gold, carbon or screen-printed conductive ink and may include an oligonucleotide or protein that will bind with the capture probe. The capture probe and the detector probes will bind with the target nucleic acid; either may be annealed across a deletion, insertion or single base alteration in the target.
2.1 Detection of Pathogens
By using specific sequences of DNA or RNA that are characteristic of target microbes, pathogens can be unambiguously identified, regardless of their cultivable states, by direct analysis of contaminated food or water samples. As used herein, xe2x80x9cmicrobesxe2x80x9d is intended to include unicellular organisms, eukaryotic cells, bacteria, viruses, cyanobacteria, fungi, yeast, molds, prions and archebacteria. Definitive data may be obtained regarding food and water quality, and the time-consuming culturing step associated with coliform counts is reduced or eliminated. In addition, distinctions can be made between different coliform bacteria, e.g. pathogenic v. nonpathogenic bacteria.
Examples of pathogens that can be detected using the invention include, but are not limited to, bacteria such as Salmonella sp., Escherichia coli, Klebsiella pneumoniae, Bacillus sp., Shigella sp., Campylobacter sp., Helicobacter pylori, Vibrio sp., Chlamydia, Giardia, parasites such as Naegleria and Acanthamoeba and viruses such as Hepatitis and poliomyelitis.
2.2 Detection of Genetic Variations
The invention may be employed to detect genetic variations associated with different disorders or diseases. Examples of diseases that can be detected include cystic fibrosis, muscular dystrophy, sickle cell anemia, phenylketonuria, thalassemia, hemophilia, a1-antitrypsin deficiency, disorders of lipoprotein metabolism and inherited forms of cancer. In addition, quantitative analysis of human genes is also desirable for analysis of amplified oncogenes (Altitalo, 1987), detection of genetic defects and in the determination of gene expression levels in tumors (Slamon et al., 1989).
Cell samples may be from biological or clinical sources which are first lysed to free nucleic acid sequences into an aqueous medium and then allowed to hybridize with capture and detector probes that incorporate the respective characteristic target nucleic acid sequences. An electrochemical signal is generated by the hybridized probes/nucleic acid sequences and detected with an electrode biosensor. In certain cases, an electron transfer mediator and an electroactive reporter group may be used to facilitate generation of an electrochemical signal.
2.3 Kits
Easy-to-use kits also form part of the invention. Such kits contain monitors, reagents and procedures that can be utilized in a clinical or research setting or adapted for either the field laboratory or on-site use. In particular, kits comprising the disclosed biosensor or biosensor array, or an apparatus comprising the biosensor in an integrated chip form, or a system that includes any of a number of means for detecting the captured target molecule and measuring the electrochemical signal produced subsequent to target capture, along with appropriate instructions, are contemplated. Kits comprising electrodes with one or more capture or reporter probes attached to the electrode surface where the probes are hybridizable with selected nucleic acid segments, one or more detector probes capable of hybridizing with a nonselected segment of the target nucleic acid along with instructions for use of the electrode or electrodes to electrochemically detect the selected target nucleic acid are also contemplated. An electrode with a 15-25 base oligonucleotide that selectively hybrizes or anneals with a targeted nucleic acid is a preferred embodiment.
The kits can be widely employed in less technologically developed areas or countries which do not have well-equipped laboratories and at remote sites far from well-equipped laboratory facilities. The invention thus is useful in monitoring for the presence of selected nucleic acids indicative of human health concerns (pathogens, genetic defects) at small laboratories, physician""s offices, bedsides, and field locations.
2.4 Screening methods
The invention further provides a screening system for rapid detection of genetic polymorphisms and small mutations scattered throughout long coding regions of the gene. The method takes advantage of inherent differences in the melting temperature of DNA strands which are perfectly homologous and comparing these to duplexes containing mismatched deleted or inserted base pairs. Depending on the extent of homology and the length of the DNA probe, the temperature at which a target probe hybrid will melt can differ by several degrees. The ability to electrochemically differentiate the melting temperatures of sample and reference for different regions of a gene provides a method to screen for mutations or polymorphisms using probes for any stretch of DNA for which the DNA sequence is known. The disclosed electrochemical differential thermal scanning approach permits rapid detection as well as the ability to localize differences between a standard reference gene and the gene rather than require de novo determination of the complete sequence of each sample.
The screening method also takes advantage of the disclosed technology to identify candidate drugs for modulating cell function. The putative drug candidate is incubated with a cell culture selected as being of interest in some therapeutic area such as antibiotic utility. Nucleic acids are extracted from the cells after the incubation and hybridized with a selected array of nucleic acid segments prepared on the disclosed biosensor array. The selected nucleic acids are bioreporter or capture probes prepared from nucleic acids with known functions, e.g., metabolic enzyme mRNAs from a pathogen. Detection and quantitation can be performed as described by applying a pulsed electrical potential to the biosensor array. A comparison of the xe2x80x9clibraryxe2x80x9d of nucleic acids from the putative drug treated cell culture with the library obtained from a cell culture without the putative drug will be indicative of a substance that affects cell function. Generally the nucleic acids to be detected will be mRNAs. One application will be to determine the effect of potential drugs on mRNA expressed from ApoA1, p53, BRCA1 and other genes associated with cancer as well as genes associated with mutated genes. The screening method may also be of use in targeting substances to mRNAs associated with gene expression in individuals with inborn errors of metabolism.
2.5 Detection of PCR Amplified Products
In another embodiment, the disclosed invention can be coupled to polymerase chain reaction (PCR(trademark)) or other nucleic acid amplification methods. Thus, DNA from a pathogen may be amplified or the RNA from a pathogen may be reverse transcribed (RT) and then amplified or amplified directly, and the amplified product detected electrochemically in accordance with the invention. The amplification and detection may be employed to increase the concentration of a pathogen for purposes of detection, or to follow the progress of an amplification reaction.
In an illustrative example, the disclosed methods may be used to detect and quantify a double-stranded PCR(trademark) product by tagging one strand and binding that strand to a NeutrAvidin-coated biosensor, and by tagging the other strand with fluorescein and reacting that strand with an anti-fluoresein HRP conjugate. Promptly following the addition of a peroxide and a mediator such as ferrocene to the PCR(trademark) product, in an electrochemical cell in accordance with the invention, an amperometric signal is generated and may be detected.
Confirmation of the product may be accomplished by first denaturing the fluorescein labeled strand from the hybrid with base. The captured (biotin-labeled) strand may be verified by forming a second hybrid with a known fluorescein-labeled probe, followed by electrochemically detecting the hybrid.